D.H. Kim et al. / Process Biochemistry 51 (2016) 1374–1379
1375
In the present study, we validated the functions of the S.
degradans 2-40T enzymes, DEH reductase, KDG kinase, and KDPG
aldolase. Based on the in vitro activities of the enzymes, we have
proposed the complete alginate metabolic pathway in S. degradans
2-40T.
using a microplate spectrophotometer (Bio-Rad Laboratories, Rich-
mond, CA, USA). The production of KDG was confirmed by thin layer
chromatography (TLC) and gas chromatography/mass spectrome-
try (GC/MS).
To test enzyme activities of KDG kinase and KDPG aldolase in
vitro, 800 g/mL of each purified enzyme were sequentially added
to post-reaction mixtures that contained the corresponding sub-
strates for the target enzymes. For the reaction by KDG kinase,
2.5 mM of ATP was added in the reaction mixture as a phospho-
ryl donor. After incubating at 30 ◦C for 1 h, each enzymatic reaction
mixture was quenched in boiling water for 5 min. The production
of KDPG by putative KDG kinase was confirmed by TLC and GC/MS,
and the production of pyruvate by putative KDPG aldolase was con-
firmed by gas chromatography/time-of-flight mass spectrometry
(GC/TOF MS).
2. Materials and methods
2.1. Cloning
For cloning of genes encoding putative DEH reductase, KDG
kinase, and KDPG aldolase enzymes, the genomic DNA of S.
degradans 2-40T was prepared as follows: S. degradans 2-40T was
cultivated for 18 h at 30 ◦C and 200 rpm in minimal medium con-
sisting of 23 g/L Instant Ocean Sea Salt (Aquarium Systems, Mentor,
OH, USA), 50 mM Tris–HCl (pH 7.4), 2 g/L glucose, 1 g/L yeast extract,
and 0.5 g/L ammonium chloride. Genomic DNA was extracted from
the culture using DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA,
USA) following the manufacturer’s protocol.
Genes encoding putative DEH reductase (short-chain dehy-
drogenase/reductase SDR, NCBI gene ID: 3965773), KDG kinase
(2-keto-3-deoxygluconate kinase, NCBI gene ID: 3965772), and
KDPG aldolase (2-keto-3-deoxy-phosphogluconate aldolase, NCBI
gene ID: 3968651) of S. degradans 2-40T were amplified by poly-
merase chain reaction (PCR) using primers listed in Table S1. Each
PCR product was treated with BamHI and NotI restriction enzymes
and then ligated with a pET-21a plasmid treated with the same
enzymes. Escherichia coli BL21 (DE3) was transformed with each
of the resulting plasmids, and transformants were selected on
Luria-Bertani agar (LBA; BD, Sparks, MD, USA) containing 50 g/mL
ampicillin.
2.5. TLC
For quick assessment of intermediates produced in the enzy-
matic reaction mixtures above, TLC was performed as previously
described [11]. Three microliters of each reaction product was
applied to a silica gel plate 60 (Merck, Darmstadt, Germany).
Metabolites were developed using an n-butanol-acetic acid-water
(3:2:2 by volume) solution and visualized by heating the TLC plate
at 130 ◦C for 5 min using a 10% (v/v) sulfuric acid solution in ethanol.
For identification of DEH, KDG, and KDPG, each post-enzymatic
reaction mixture was dried and derivatized as previously described
[21]. Briefly, the aldehyde group of each reaction product
was derivatized by methoxyamination using 50 L of 20 mg/mL
methoxyamine hydrochloride in pyridine (Sigma-Aldrich, St. Louis,
MO, USA) at 75 ◦C 30 min. The volatility of the sample was increased
by adding 80 L of N-methyl-N-(trimethylsilyl)tri-fluoroacetamide
(Sigma-Aldrich, St. Louis, MO, USA). The derivatized samples (1 L)
were analyzed with an Agilent 7890A GC/5975C MSD system (Agi-
lent Technologies, Wilmington, DE, USA) equipped with a DB5-MS
column (0.25 mm × 30 m, 0.25 m film thickness; Agilent Tech-
nologies). The temperature of the GC oven was programmed as
follows: 100 ◦C for 3.5 min, increased to 160 ◦C by 15 ◦C/min and
held for 20 min, increased to 200 ◦C by 20 ◦C/min and held for
20 min, and increased to 280 ◦C by 20 ◦C/min and held for 5 min.
Ionization was achieved by electron impact at 70 eV, and the tem-
perature of the ion source was 230 ◦C. The mass spectra were
recorded in the range of 50–500 m/z [21].
2.2. Enzyme production and purification
For the production of target enzymes, E. coli transformants were
cultivated in LBA at 37 ◦C and 180 rpm. When the absorbance of the
culture at 600 nm (A600) reached 0.5, 0.1 mM IPTG was added, and
the culture was further incubated for 16 h at 16 ◦C. Cells were col-
lected and disrupted by sonication (Branson, Danbury, CT, USA).
After centrifugation of the lysate at 16,000 × g for 60 min, soluble
supernatant was applied to a His-Trap column (GE Healthcare, Pis-
cataway, NJ, USA), and proteins were eluted in Tris–HCl (pH 7).
The enzyme eluent was concentrated using an Amicon tube (Milli-
pore, Billerica, MA, USA). The concentrated enzyme solutions were
analyzed with a bicinchoninic acid (BCA) protein assay (Pierce,
Rockford, IL, USA) and 12% (w/v) SDS-PAGE gel electrophoresis to
For analysis of pyruvate, an Agilent 7890A GC (Hewlett-Packard,
Atlanta, GA, USA) coupled to a Pegasus HT TOF MS (Leco, St. Joseph,
MI, USA) was used. This GC/TOF MS was equipped with an RTX-5Sil
MS capillary column (Restek, Bellefonte, PA, USA) and an additional
2.3. Substrate preparation
As a substrate for the in vitro assay of DEH reductase activity,
alginate solution (2% w/v, Sigma-Aldrich, St. Louis, MO, USA) was
treated sequentially with recombinant endo-type alginate lyase
(Alg7D originating from S. degradans 2-40T) and exo-type alginate
lyase (Alg17C originating from S. degradans 2-40T) to produce algi-
nate oligosaccharides and DEH, respectively [11,12]. The reaction
solution then underwent Bio-Gel P-2 size exclusion column chro-
matography to obtain purified DEH.
4-Deoxy-L-erytro-5-hexoseulose uronate
(MW 176)
35E+05
30E+05
73.1
CHO
l
OHCH
l
OHCH
l
290.1
25E+05
20E+05
15E+05
10E+05
5E+05
0
CH2
l
C=O
l
COOH
147.1
217.1
200.1 258.1
133.1
2.4. Enzyme assay of putative metabolic enzymes
50 100 150 200 250 300 350 400 450 500
For the in vitro analysis of DEH reductase activity, 1 mL reaction
mixture was prepared with 20 mM Tris–HCl (pH 7), 800 g/mL of
purified enzyme, and either 1 mM NADH or 1 mM NADPH. Reduc-
tions in the absorbance at 340 nm was monitored for 40 min at 30 ◦C
m/z
Fig. 1. Mass spectrum of the substrate, 4-deoxy-l-erythro-5-hexoseulose urinate
(DEH) prepared in this study.
Kim, Do Hyoung
